~ 384 ~

Journal of Medicinal Plants Studies 2017; 5(3): 384-391

ISSN (E): 2320-3862

ISSN (P): 2394-0530

NAAS Rating 2017: 3.53

JMPS 2017; 5(3): 384-391

© 2017 JMPS

Received: 11-03-2017

Accepted: 13-04-2017

Pratibha Singh

Sophisticated Analytical

Instrument Facility, CSIR-

Central Drug Research Institute,

Lucknow, Uttar Pradesh, India

Vikas Bajpai

a) Sophisticated Analytical

Instrument Facility, CSIR-

Central Drug Research Institute,

Lucknow, Uttar Pradesh, India

b) Academy of Scientific and

Innovative Research (AcSIR),

New Delhi, India

Sunil Kumar

Sophisticated Analytical

Instrument Facility, CSIR-

Central Drug Research Institute,

Lucknow, Uttar Pradesh, India

Mukesh Srivastava

a) Academy of Scientific and

Innovative Research (AcSIR),

New Delhi-110025, India

b) Biometry and Statistics

Division, CSIR-Central Drug

Research Institute, Lucknow -

226031, Uttar Pradesh, India

Brijesh Kumar

a) Sophisticated Analytical

Instrument Facility, CSIR-

Central Drug Research Institute,

Lucknow, Uttar Pradesh, India

b) Academy of Scientific and

Innovative Research (AcSIR),

New Delhi, India

Correspondence

Brijesh Kumar

Professor (AcSIR) & Senior

Principal Scientist, Sophisticated

Analytical Instrument Facility,

CSIR-Central Drug Research

Institute, Lucknow.

Uttar Pradesh, India

Metabolic profiling and discrimination of

Cymbopogon species using direct analysis real

time mass spectrometry and principal component

analysis

Pratibha Singh, Vikas Bajpai, Sunil Kumar, Mukesh Srivastava and

Brijesh Kumar

Abstract Several Cymbopogon species such as C. citratus, C. flexuosus, C. nardus and C. khasianus×C. pendulus

are extensively used for flavors, fragrance and as folk medicines worldwide due to their volatile essential

oils (VEOs). Direct analysis in real time mass spectrometry (DART-MS) method was developed for

identification of VEOs from the intact plant parts of ten Cymbopogon species. Total sixteen compounds

including VEOs, phenolics and flavonoids were tentatively identified on the basis of their exact mass

measurement and molecular formula using DART-MS chemical fingerprint of C. citratus, C. citronella,

C. flexuosus, C. pendulus, C. commutatus, Cymbopogon. jwarancusa, C. nardus, C. khasianus, C.

jwarancusa× C. nardus and C. khasianus× C. pendulus. DART-MS data was analyzed by principal

component analysis and 19 marker peaks were identified which can discriminate among these selected

Cymbopogon species. C. citratus, C. jwarancusa, C. commutatus, C. khasianus and C. jwarancusa×C.

nardus were approximately overlapping each other while C. flexuosus, C. nardus and C. khasianus×C.

pendulus were closer to each other whereas C. pendulus and C. citronella were much apart from all the

species. The identified marker peaks could be used for authentication, discrimination and quality control

of Cymbopogon species.

Keywords: DART-MS, Volatile essential oils, Cymbopogon species, PCA

1. Introduction Genus Cymbopogon (Family; Poaceae) is well known tropical perennial shrub [1-2]. The plants

of this family are distributed worldwide [1-2]. Cymbopogon species has been reported to possess

various pharmacological activities such as analgesic [2], antibacterial [3], anticarcinogenic [3],

cardioprotective [3], antifungal [3], anti-inflammatory [4], antileishmanial [5], antioxidant [6],

antiprotozoal [7], antipyretic [8], antirheumatic [9], antitrypanosomal [10], antiseptic [11],

antispasmodic [12], antitussive [12] and antiviral [13] activities. They also possess some

pharmacological properties such as diuretic and sedative [14]. C. citratus belonging to the genus

Cymbopogon has traditionally been used for the treatment of anxiety, diabetes, dyslipidemia,

fever, flu, gastrointestinal disturbances, malaria, and pneumonia [15]. It has also been used to

inhibit platelet aggregation [16]. Phytochemicals such as volatile essential oils (VEOs), tannins,

saponins, flavonoids, alkaloid and terpenoids are reported in Cymbopogon species [17]. The

essential oil, Cryptomeridiol shows antispasmodic activity [18]. Myrcene, citral, cis-linalool

oxide and cryptomeridiol are the major commercially available component of VEOs from

Cymbopogon species (commonly from Cymbopogon citratus, Cymbopogon flexuosus,

Cymbopogon pendulus, Cymbopogon commutatus, Cymbopogon jwarancusa, Cymbopogon

nardus, Cymbopogon khasianus, Cymbopogon jwarancusa× Cymbopogon nardus and

Cymbopogon khasianus× Cymbopogon pendulus) and are used as fragrance and flavour in

pharmaceutical industries [19], aromatherapy [20] and chemotherapy [21].

Various analytical methods such as electrospray ionization mass spectrometry (ESI-MS) [22],

fourier transform infra-red spectroscopy (FTIR) [23], nuclear magnetic resonance (NMR) [24]

were used to analyze the cryptomeridiol a major component of VEOs of Cymbopogon species.

The other essential oils component such as citronellol and citral were also studied by high

performance thin layer chromatography (HPTLC) [25] and quantified by using a high

performance liquid chromatography (HPLC) method [26] in Cymbopogon citratus.

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Journal of Medicinal Plants Studies

Due to the volatile nature, the analysis of essential oils from

Cymbopogon species is mainly reported by gas

chromatography-mass spectrometry (GC-MS).[27] However,

the HPLC ESI-MS based identification and characterization

of flavonoids in C. citratus leaves has been also reported.[22,28]

These methods are tedious and need time-consuming sample

preparation and chromatographic separation steps.

Direct analysis in real time (DART) mass spectrometry is an

ionization technique which ionizes samples under ambient

conditions [29]. It can ionize solid, liquid and gas samples

directly without any samples preparation [30]. This technique

has been successfully used for identification of pesticides,

explosives on solid surfaces and in liquids, chemical warfare

agents in solvents, food packaging additives, flavored

contents of food, contaminant in soil, cocaine in urine with

vast range of applications in forensics [31, 32]. Recently DART-

MS followed by multivariate analysis were also used for

discrimination of plant species [33-37] and cultivars [38],

authentication of animal fats [39] detection of adulteration and

geographical variation [39-40].

This manuscript aimed to develop an efficient DART MS

method for identification of VEOs from C. citratus, C.

citronella, C. flexuosus, C. pendulus, C. commutatus, C.

jwarancusa, C. nardus, C. khasianus, C. jwarancusa×C.

nardus and C. khasianus×C. pendulus. The PCA was used to

discriminate the selected Cymbopogon species and

identification of marker peaks which can be used for

authentication and quality control of this plant.

2. Materials and Methods

2.1 Materials

The leaves of C. citratus, C. citronella, C. flexuosus, C.

pendulus, C. commutatus, C. jwarancusa, C. nardus, C.

khasianus, C. jwarancusa×C. nardus and C. khasianus×C.

pendulus were collected in June 2015 from CSIR-India

Institute of Integrated Medicine (CSIR-IIIM), Jammu.

Voucher specimens of C. citratus-RRL-52923, C. citronella-

RRL-52926, C. flexuosus-RRL-52925, C. pendulus- RRL-

52932, C. commutatus- RRL-52919, C. jwarancusa-RRL-

52927, C. nardus-RRL-52924, C. khasianus-RRL-52930, C.

jwarancusa×C. nardus-RRL-52931 and C. khasianus×C.

pendulus-RRL-52928 were deposited in medicinal plant

herbarium of CSIR-IIIM, Jammu. Standard samples of p-

coumaric acid, Kaempferol, Cholorogenic acid and Orientin

were purchased from Sigma Aldrich. The leaf samples were

thoroughly washed with tap water followed by distilled water

to remove foreign particles from its surface and dried at room

temperature (approximately 26-28°C).

2.2 DART MS operating parameters

The mass spectrometer used was a JMS-T100LC, Accu TOF

atmospheric pressure ionization time-of-flight mass

spectrometer (Jeol, Tokyo, Japan) fitted with a DART ion

source. The mass spectrometer was operated in positive-ion

mode with a resolving power of 6000 (full-width at half-

maxima). The orifice 1 potential was set to 28 V, ring lens

and orifice 2 potentials were set to 13 and 5 V, respectively.

Orifice 1 was set at 100°C and RF ion guide potential at 300

V. The DART ion source was operated with helium gas

flowing at approximately 4.0 L/min and gas heater was set at

300°C. The potential on the discharge needle electrode of the

DART source was set to 3000 V, electrode 1 at 100 V and the

grid at 250 V. Data acquisition was from m/z 50 to 1050. All

the leaf samples were analyzed in 15 repeats to check the

reproducibility of spectra. Mass calibration was accomplished

by including a mass spectrum of neat polyethylene glycol

(PEG) (mixture of PEG 200 and PEG 400) in the data file.

The mass calibration was accurate to within ±0.002 u. Using

the Mass Centre software, the elemental composition were

determined on selected peaks.

2.3 Statistical analysis

Principal component analysis (PCA) was performed with the

STATISTICA software, windows version 7.0 (Stat Soft, Inc.,

USA). Data for PCA analysis was extracted from DART-MS

spectra of fifteen repeats of each sample. All ions having ≥5%

peak intensity were selected for statistical analysis.

3. Results and Discussion

3.1 Screening of phytochemicals in Cymbopogon species

Comparative DART-MS fingerprint spectra of the

Cymbopogon species are shown in Fig. 1. All spectra showed

common peaks (m/z) with different relative abundance.

Sixteen phytochemicals were tentatively identified based on

their exact mass, molecular formula and literature reports [27,

41-42] as shown in (Table 1) and structure of all the identified

phytochemicals are given in Fig 2. The peaks at m/z 137.1337

(C10H16), 153.1263 (C10H16O), 157.1596 (C10H20O), 170.1374

(C10H18O2) and 205.1957 (C15H24) were identified as myrcene

(2), citral (3), citronellol (5), cis-linalool oxide (7) and γ-1-

cadinene (9) respectively. All the identified compounds were

again confirmed by their exact mass and fragmentation

patterns using HPLC-ESI-QTOF-MS/MS study.

Fragmentation patterns of compounds p-coumaric acid (6),

Cis-Linalool oxide (7), Caffeic acid (8), Caryophyllene oxide

(10), Cryptomeridiol (12), Kaempferol (13), Cymbodiacetal

(14) Cholorogenic acid (15) and Orientin (16) are also

compared from literature and/or standard compounds for

authentication Fig. S1 A and B, Figure. S2 and Table S1 and

S2. Details of experimental method and figures are provided

in supplementary.

3.2 Comparison of DART-MS fingerprint of Cymbopogon

species All these VEOs were detected in relatively high abundance in

the leaves of Cymbopogon species. Myrcene (2) m/z 137.1337

(C10H16) was detected in high abundance in C. citratus, C.

citronella, C. commutatus, C. jwarancusa, C. nardus, C.

khasianus and C. jwarancusa×C. nardus while citral (3) m/z

153.1263 (C10H16O) was found abundant in all the species of

Cymbopogon except C. citronella. Similarly, citronellol (5)

m/z 157.1596 (C10H20O) was identified in high abundance in

the leaves of C. citratus, C. citronella and C. flexuosus while

γ-1-cadinene (9) m/z 205.1957 (C15H24) was detected high

abundance in the leaves of C. citratus, C. citronella and C.

jwarancusa×C. nardus. The peak at m/z 223.2068 (C15H26O)

identified as β-eudesmol (11) was found relatively high in C.

citratus followed by C. commutatus and C. khasianus×C.

pendulus. The peak at m/z 241.2172 (C15H28O2), identified as

cryptomeridiol (12), was found relatively in low abundance in

C. citratus followed by C. commutatus and C. khasianus×C.

pendulus. The peak at m/z 449.1088 (C21H20O11) was

identified as Orientin (16) with high intensity in C. citratus,

C. citronella, C. pendulus and C. commutatus. All the

identified phytochemicals were present in C. citratus except

p-coumaric acid (6) at m/z 165.0561 (C9H8O3) and citral (3) at

m/z 153.1263 (C10H16O) was detected in all the Cymbopogon

species except C. citronella Table 1.

The DART MS spectra revealed the variation in the relative

intensities of some of the most common essential oils in the

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Journal of Medicinal Plants Studies

leaves of studied species Fig. 3. It was obtained as the ratio of

the expression of the peak to the sum of all the expressions

within the spectra ranging from m/z 95-550 as shown in Fig.

1. All the ions with a relative intensity above 5% were taken

and compared on the basis of these relative intensities. Fifteen

repeats were carried out for each sample and the averaged

result was utilized for analysis. The results indicated

significant variations of bioactive compounds among the

leaves of all the ten Cymbopogon species Fig. 3.

Approximately similar relative content of myrcene (2) at m/z

137.1337 (C10H16) was detected in C. khasianus and C.

jwarancusa×C. nardus followed by C. commutatus and C.

citronella while it was found in relatively low abundance in

C. jwarancusa and C. nardus. Similarly, citral (3) at m/z

153.1263 (C10H16O) was detected approximately in same

abundance in C. khasianus×C. pendulus, C. citratus, C.

flexuosus, C. pendulus, C. jwarancusa and C. nardus while it

was detected relatively in low abundance in C. khasianus and

C. jwarancusa×C. nardus. Whereas cis-linalool oxide (7) at

m/z 170.1374 (C10H18O2) was abundant in C. citronella, C.

pendulus, C. jwarancusa and C. citratus. γ-1-cadinene (9) at

m/z 205.1957 (C15H24) and orientin (16) at m/z 449.1088

(C21H20O11) were abundant in C. citronella and C.

commutatus respectively.

3.3 Discrimination of Cymbopogon species using principle

component analysis PCA is an unsupervised procedure that determines the

directions of the largest variations in the data set and the data

are generally presented as a two dimensional plot (score plot)

where the coordinate axis represents the directions of the two

largest variations [33-34]. DART-MS data combined with

principal component analysis (PCA) served as an efficient and

powerful tool to identify the chemical markers and to

discriminate among Cymbopogon species [33-34]. The DART-

MS data from fifteen repeats of each species (C. citratus, C.

commutatus, C. flexceosus, C. pendulus, C. jwarancusa, C.

citronella, C. nardus, C. khasianus, C. jwarancusa×C. nardus

and C. khasianus×C. pendulus) were subjected to PCA.

The first two principal components PC1 and PC2 hold

31.76% and 30.12% respectively of the total variability. Thus,

the PCs were able to explain 61.88% of the total variability.

To obtain the best expression some peaks having low scores

were dropped to get the best possible results. Finally, the first

two principal components PC1 and PC2 hold 32.48% and

31.65% respectively of the total variability on the basis of 19

peaks at m/z 95.1022, 123.1415, 135.1439, 151.1754,

153.1263 (Citral), 155.1407 (citronellal), 157.1596

(citronellol), 169.1555, 170.1374 (cis-Linalool oxide),

183.1255, 200.1608, 205.1957 (γ-1-Cadinene), 221.1965

(Caryophyllene oxide), 228.2416, 237.2230, 305.3056,

312.3203, 409.4548 and 449.1088 (Orientin) Fig. 4A. Out of

36 peaks only 19 peaks showed 64.13% total variance. Peak

at m/z 95.1022 (32.48%) gave a higher contribution for

discrimination followed by peak at m/z 123.1415 (31.65%).

The PCA discriminated all the ten Cymbopogon species in to

four categories. C. citronella and C. pendulus were standing

isolated in the bi-plot which indicated these two species have

entirely different pattern than the rest species studied. The

remaining eight species were further divided in two sub

groups. In the first sub group the species were C. nardus, C.

flexuosus and C. khasianus×C. pendulus, this sub groups has

peaks at m/z 95.1022, 135.1439, 153.0757 and 305.3056

which were detected in all the three species of the group.

In the second sub group there were five species namely C.

khasianus, C. jwarancusa×C. nardus, C. citratus, C.

jwarancusa and C. commutatus which have close similarities.

This group was dominated by peaks at m/z 183.1255 and

200.1608 which was detected in all the five species except C.

jwarancusa×C. nardus in which peak at m/z 200.1608 was

not detected.

The major difference in these two sub groups was presence

and absence of peaks at m/z 95.1022, 153.1263 and 305.3056.

The species C. citronella was dominated by the high intensity

of peak at m/z 205.1957 which was not detected in C.

pendulus. The abundance of myrcene (m/z 137.1337) and

citral (m/z 153.1263) were detected approximately five and

two fold higher in C. jwarancusa×C. nardus and C.

khasianas×C. pendulus compared to their parents, C.

jwarancusa, C. nardus and C. khasianus, C. pendulus

respectively. Hence these hybrid species may be selected for

the isolation of myrecene (2) and citral (3) respectively. It is

evident from this study that PCA effectively served the

desired purpose.

3.4 Comparative chemical fingerprint of C. jwarancusa, C.

nardus and C. jwarancusa × C. nardus Out of 36 studied peaks, 22 peaks were present in either C.

jwarancusa and C. nardus or its hybrid (C. jwarancusa×C.

nardus). The distributions of these peaks are shown by the

help of Venn diagram in Fig. 5A. Seven peaks were detected

(at m/z 95.1022, 135.1439, 167.1392, 169.1555, 284.2937,

287.2944 and 305.3056) in C. nardus whereas two peaks (at

m/z 110.0992 and 200.1608) were detected only in C.

jwarancusa while only one peak at m/z 170.1374 was detected

in both the species but absent in C. jwarancusa×C. nardus.

Total of twelve abundant peaks (at m/z 127.1131, 137.1337,

153.1263, 154.1919, 183.1172, 205.1957, 273.3163,

279.2169, 393.5278, 409.4548, 459.5511 and 503.1269) were

detected in hybrid. Out of twelve peaks only 6 peaks (at m/z

183.1172, 279.2169, 393.5278, 409.4548, 459.5511 and

503.1269) were common with C. jwarancusa and C.

jwarancusa×C. nardus. Only two peaks (at m/z 137.1337 and

153.1263) were detected in all the three species. The

remaining four peaks (at m/z 127.1131, 154.1919, 205.1957

and 273.3163) were detected only in C. jwarancusa×C.

nardus which were completely absent in C. jwarancusa and

C. nardus.

3.5 Comparative Chemical fingerprint of C. khasianus, C.

pendulus and C. khasianus × C. pendulus

The DART-MS analysis of C. khasianus, C. pendulus and its

hybrid (C. khasianus×C. pendulus) produced total 22 peaks.

The distributions of these peaks are shown by the help of

Venn diagram in Fig. 5 B. Four peaks (at m/z 137.1337,

273.363, 296.1506 and 445.3061) were detected only in C.

khasianus. Six peaks (at m/z 123.1415, 151.1754, 228.2416,

312.3203, 409.4548 and 449.1088) were detected only in C.

pendulus while only two peaks (at m/z 183.1172 and

200.1608) were detected in both the species but absent in C.

khasianus×C. pendulus. Total ten abundant peaks (at m/z

95.1022, 104.1123, 110.0992, 135.1439, 153.1263, 170.1374,

279.2169, 305.3056, 393.5278 and 503.1269) were detected

in C. khasianus×C. pendulus. Out of these ten peaks only two

peaks (at m/z 135.1439 and 170.1374) were common with C.

pendulus and three peaks (at m/z 110.0992, 153.1263 and

279.2169) were detected in all the three species. The

remaining five peaks (at m/z 95.1022, 104.1123, 305.3056,

393.393.5278 and 503.1269) were detected in the C.

khasianus×C. pendulus but completely absent in C. khasianus

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Journal of Medicinal Plants Studies

and C. pendulus. There was no common peak in C. khasianus

and C. khasianus×C. pendulus individually.

Only two common peaks (at m/z 137.1337 and 153.1263)

were detected in C. jwarancusa, C. nardus and C.

jwarancusa×C. nardus. Similarly three common peaks (at m/z

110.0992, 153.0757 and 279.2169) were identified in C.

khasianus, C. pendulus and C. khasianus×C. pendulus.

4. Conclusions The volatile essential oils were successfully analyzed and

identified in ten Cymbopogon species by DART-MS analysis.

The 16 bioactive compounds including eleven VEOs

components such as 6-Methylhept-5-en-2-one (1), myrcene

(2), citral (3), citronellal (4), citronellol (5), cis-Linalool oxide

(7), γ-1-Cadinene (9), caryophyllene oxide (10), β-Eudesmol

(11), cryptomeridiol (12) and cymbodiacetal (14), three

phenolics such as p-coumaric acid (6), caffeic acid (8) and

chlorogenic acid (15) and two flavonoids, kaempferol (13)

and orientin (16) were successfully identified in the

Cymbopogon species. Present study provided information

which may help in selection of Cymbopogon species on the

basis of their relative abundance of bioactive or commercially

useful VEOs components such as myrcene (2), citral (3),

citronellal (4), citronellol (5). DART-MS followed by PCA

showed the similarity and dissimilarity among the

Cymbopogon species (C. citratus, C. citronella, C. flexuosus,

C. pendulus C. commutatus, C. nardus, C. jwarancusa, C.

khasianus, C. jwarancusa×C. nardus and C. khasianus×C.

pendulus). This is first study by DART-MS for identification

of volatile essential oils and discrimination of Cymbopogon

species by PCA.

Fig 1: DART-MS fingerprint of ten Cymbopogon species (C. citratus, C. citronella, C. flexuosus, C. pendulus, C. commutatus, C. jwarancusa,

C. nardus, C. khasianus, C. jwarancusa×C. nardus and C. khasianus×C. pendulus).

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Journal of Medicinal Plants Studies

Fig 2: Chemical structures of identified compounds (1-16) in Cymbopogon species

Fig 3: Relative intensity of bioactive compounds in Cymbopogon species.

Fig. 4: (A) PC1 vs PC2 plot showing discrimination among the leaves of Cymbopogon species. (B) PC1 vs PC2 score plot showing loading of

variables.

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Journal of Medicinal Plants Studies

Fig. 5: (A) Distribution of peaks (m/z) among the C. jwarancusa, C. nardus and C. jwarancusa×C. nardus (A) and C. khasianus, C. pendulus

and C. khasianus×C. pendulus (B).

Table 1: DART-MS mass measurements of phytochemicals in leaves of Cymbopogon species

S.

No Compounds name

Calculated mass

(m/z)

Measured

Mass (m/z)

Molecular

formula

Error

(Δmmu)

Distribution

Cc Cct Cf Cp Ccm Cj Cn Ck Cj×n Ck×p

1. 6-Methylhept-5-en-2-

one 127.1123 127.1131 C8H14O 0.81 + - - - - - - - + -

2. Myrcene 137.1330 137.1337 C10H16 -0.71 + + - - + + + + + -

3. Citral 153.1279 153.1263 C10H16O 0.61 + - + + + + + + + +

4. Citronellal 155.1436 155.1407 C10H18O -2.90 + + - - - - - - - -

5. Citronellol 157.1592 157.1596 C10H20O -0.40 + + + - - - - - - -

6. p-Coumaric acid 165.0552 165.0561 C9H8O3 0.91 - - + - - - + - - -

7. cis-Linalool oxide 170.1385 170.1374 C10H18O2 1.08 + - - + + + + - - +

8. Caffeic acid 181.0501 181.0509 C9H8O4 -0.61 + - - - - - - - - -

9. γ-1-Cadinene 205.1956 205.1957 C15H24 1.37 + + - - - - - - + -

10. Caryophyllene oxide 221.1905 221.1965 C15H24O 3.20 + + - - - - - - - -

11. β-Eudesmol 223.2062 223.2068 C15H26O -1.81 + - - - + - - - - +

12. Cryptomeridiol 241.2168 241.2172 C15H28O2 3.69 + - - - + - - - - +

13. Kaempferol 287.0556 287.0575 C15H10O6 -1.27 + - - - - - + - - -

14. Cymbodiacetal 335.2222 335.2226 C20H30O4 -0.40 + - - - + - - - - -

15. Chlorogenic acid 355.1029 355.1028 C16H18O9 0.25 + - - - + + - - - +

16. Orientin 449.1084 449.1088 C21H20O11 1.10 + + - + + + - - - - a(+): detected, (-): not detected, Cc: C. citratus, Ct: C. citronella, Cf: C. flexuosus, Cp: C. pendulus, Ccm: C. commutatus, Cj: C. jwarancusa,

Cn: C. nardus, Ck: C. khasianus, Cj×n: C. jwarancusa×C. nardus and Ck×p: C. khasianus×C. pendulus

5. Acknowledgements Grateful acknowledgement is made to the Sophisticated

Analytical Instrument Facility (SAIF), Central Drug Research

Institute, Lucknow, where the mass spectral studies were

carried out. Authors are also thankful to Bikarma Singh,

Scientist Biodiversity and Applied Botany Division, CSIR-

Indian Institute of Medicine, Jammu India. Pratibha Singh is

thankful to MoES for grant.

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